Mapping of Cerebral Blood Flow Changes during ... - SAGE Journals

Journal of Cerebral Blood Flow and Metabolism 15:259-269 © 1995 The International Society of Cerebral Blood Flow and Metabolism Published by Raven Press, Ltd., New York

Mapping of Cerebral Blood Flow Changes During Audiogenic Seizures in Wistar Rats: Effect of Kindling

Astrid Nehlig, Marguerite Vergnes, Edouard Hirsch, *Sylvette Boyet, *Violette Koziel, and Christian Marescaux INSERM U 398, Hopitai Civil, Clinique Neurologique, Strasbourg, and *INSERM U 272, Nancy, France

Summary: The quantitative autoradiographic [14C]iodo antipyrine technique was applied to the measurement of rates of local cerebral blood flow (LCBF) during audio genic seizures in Wistar AS rats belonging to a genetic strain selected at the Centre de Neurochimie (Strasbourg, France) for their sensitivity to sound. Seizures were elic ited in naive rats never exposed to sound (single audio genic seizures) or in rats previously exposed to l�O seizure-inducing sound stimulations until generalization of the seizure to forebrain areas (referred to as "kindled animals"). During single audiogenic seizures, rates of LCBF increased over control values in all areas but the genu of the corpus callosum. The highest increases in LCBF (180-388%) were recorded in the inferior and su perior colliculus, reticular formation, monoaminergic cell groupings, especially the substantia nigra, posterior veg etative nuclei, and many thalamic and hypothalamic re gions. The lowest increases were seen in forebrain limbic regions and cortical areas. In kindled animals, LCBF rates increased over control levels in 67 areas of the 75

studied. LCBF increases were generally of a lower am plitude in kindled than in naive rats. Differences between the two groups of seizing rats were located mostly in

In the breeding colony of our laboratory (Centre de Neurochimie, Strasbourg, France), we have se lected a strain of Wistar rats susceptible to audio genic seizures (Wistar AS) (Marescaux et aI., 1987). In these rats, the exposure to an intense sound stim ulus induces an epileptic seizure characterized by one or two wild running episodes lasting for about 10-30 s, followed by a tonic phase with dorsal hy perextension, the forelimbs stretched out forward and the hindlimbs flexed. This seizure is followed

by a prolonged catatonic state. During the wild run ning episodes, no paraxosmal activity appears on the electroencephalographic (EEG) recording, while the EEG transiently flattens during the tonic phase (Marescaux et aI., 1987; Kiessman et aI., 1988), as described previously in audiogenic mice (Maxson and Cowen, 1973; Willot, 1977) and rats (Kruschinsky et aI., 1970). In Wistar AS rats, after 10-40 sound exposures, facial and forelimb clonus with rearing and falling and/or tonic-clonic seizures progressively appear. During these myoclonic or tonic-clonic seizures, on the EEG rhythmic spikes, polyspikes and spikes and waves of a high ampli tude appear for 40- 120 s. Thus, the progressive and permanent modification of behavioral and EEG pat· terns occurring when audiogenic seizures are re peated suggests that kindling has developed (Mares caux et aI., 1987; Vergnes et aI., 1987; Kiessman et

brain-stem regions, mainly the inferior colliculus, reticu lar formation, substantia nigra, and posterior vegetative nuclei. Conversely, rates of LCBF were similar in fore brain areas of naive and kindled animals. In conclusion, the present data show that there is a good correlation between the structures known to be involved in the ex pression of audiogenic seizures (inferior col\iculus, retic ular formation, substantia nigra mainly) and the large in crease in LCBF during single audiogenic seizures, while rates of LCBF increase to a lesser extent in forebrain areas not involved in this type of seizures. The circula tory adaptation to kindled seizures is rather a decreased response in brain-stem regions and no change in the fore brain, although the kindling process induces a generaliza tion of the seizure from brain-stem to anterior regions. Key Words: Audiogenic seizures-[14C]Iodoantipyrine Kindling-Local cerebral blood flow.

Received March 21, 1994; final revision June 14, 1994; ac cepted July 9, 1994. Address correspondence and reprint requests to Dr. A. Nehlig, INSERM U 398, Laboratoire de Physiologie, Faculte de Medecine, 9 Avenue de la Foret de Haye, B.P. 184, 54505 Van doeuvre-Les-Nancy-Cedex, France. Abbreviations used: GEPR, genetically epilepsy-prone rat; [14C)IAP, 4-iodo-N-[methyl-14C]iodoantipyrine; LCBF, local ce rebral blood flow; NE, nonepileptic.

259

260

A. NEHLIG ET AL.

al. , 1988). These seizures showing a generalization from brain-stem to forebrain areas are referred to as kindled audiogenic seizures. A typical EEG record ing of a single audiogenic seizure in a naive rat and of a kindled audiogenic seizure is shown in Fig. 1. Audiogenic seizures are "brain-stem" seizures, related to genetically determined or acquired dys functions of the auditory pathways (Faingold et al. , 1986; Millan, 1988; Jobe et al. , 1991). Due to degen en(tion of cochlear hairs, strong acoustic stimuli in duce an abnormal response within the inferior col liculus accompanied by wild running episodes (Portman et al. , 197 1; Penny et al. , 1983). Spread of the abnormal response epileptic activity to the su perior colliculus, the brain-stem reticular forma tion, and the substantia nigra results in the tonic phase (Kesner, 1966; Willot and Lu, 1980; Jobe, 198 1; McCown et al. , 1984; Browning et al. , 1985). The hippocampus, amygdala, and most forebrain structures are not implicated in the audiogenic sei zures network (Browning, 1986; Faingold, 1988; Millan, 1988). Conversely, repeated auditory stim ulations cause a progressive propagation of the ep ileptic discharge and the seizure progressively ex tends from brainstem to forebrain, such as limbic structures and neocortices (Savage et al., 1986; Marescaux et al. , 1987; Jobe et al., 1991).

SAS

+

WR

TS

, _. NJk�'wr�foJv'-"""_�

�

, .... .... .... , .... . ' . . ' ,,,'0"-.

' _ ..... -.... .

CS

�;'ii��-��f�/tl�il�tf��I�I\Wt�ttll\\\��l��I/I I)1n FIG. 1. Typical examples of cortical EEG recordings corre sponding to a single audiogenic seizure (SAS) and to a kin dled audiogenic seizure (KAS). The EEG was recorded through frontoparietal epidural electrodes (Marescaux et ai., 1987). The arrow indicates the onset of sound. WR, wild run ning; TS, tonic seizures; CS, tonic-clonic seizures. Calibra tion bars: 1 s, 200 mY.

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Cereb Blood Flow Metab, Vol. 15, No.2, 1995

There are no quantitative data available on the mapping of cerebral metabolic or circulatory func tional activity during sound-induced seizures. Only Miller et al. ( 1993) explored the changes in cerebral energy metabolism in genetically epilepsy-prone rats (GEPRs) using 5-min 2- [ 14C]deoxyglucose qual itative studies. Therefore, in the present study, we investigated the changes in the rates of local cere bral blood flow (LCBF) audiogenic seizures in na ive and kindled Wistar AS, using the quantitative autoradiographic 4-iodo-N-[methyl- 14C]iodoantipy rine ([ 14C]IAP) technique of Sakurada et al. ( 1978), which allows the measurement of regional CBF over very short periods of time, ranging from 20 to 90 s. MATERIALS AND METHODS Animals Adult Wistar AS rats from our breeding colony of audiogenic animals and control nonepileptic (NE) rats from a strain insensitive to sound were used for the study. Animals were constantly maintained un der standard laboratory conditions on a 12/ 12-h light/dark cycle (lights on at 0600 h). The experi ments were performed on a total number of 24 rats. Eight NE control animals exposed to the auditory stimulus did not show any behavioral modification indicative of a seizure. Nine naive Wistar AS rats, submitted for the first time to the acoustic stimulus, exhibited a typical single audiogenic seizure, with wild running followed by a tonic phase. Seven Wistar AS rats were exposed to 20-40 sound stim ulations extending over a 1- to 2-month period. At the end of this period, they displayed fully kindled myoclonic and/or tonic-clonic seizures in response to the acoustic stimulus. A femoral artery and vein were catheterized with polyethylene catheters (Biotrol No. 2; 0. 38-mm I. D. , 1. 09-mm O. D. ; Nogent-sur-Marne, France) under light halothane anesthesia. Both catheters were threaded under the skin, up to the back of the hindleg, to allow free access to the catheters with out disturbing the rat's movements. The animals were allowed to recover from surgery in their home cages for at least 3 h before the onset of the exper iment. All animal experimentation was conducted in conformity with the "Guiding Principles for Re search Involving Animals and Human Beings. " Measurement of LCBF LCBF rates were measured by means of the 1 4 [ C]IAP method described by Sakurada et al. ( 1978). Just before sound exposure, rats were taped at the abdominal level on rat-shaped wooden

261

LCBF AND AUDIOGENIC SEIZURES

boards. This procedure allowed easy access to cath eters, partial immobilization of the animals for blood sampling, and free movements of the four limbs during the wild running and tonic-clonic phases. Both control and audiogenic rats were ex posed to the same sound stimulus generated by an ultrasonicator ( 120 dB, 10,000-20,000 Hz). lAP in fusion was initiated after the beginning of the first running phase in audiogenic rats and after approx imately the same period of exposure to sound (about 20 s) in controls. The sound was then main tained at the same intensity level during the 60-s period of blood flow measurement. The e4C]lAP (sp ac, 1.85-2. 2 GBq/mmol; Amer sham, Little Chalfont, Buckinghamshire, UK) was injected into the animals through the femoral vein at a concentration of 925 Bq/ml. The period of mea surement of LCBF was approximately I min in du ration, during which about 1. 5 ml of the e4C]lAP solution was administered to the rats. The intrave nous infusion was conducted at a progressively in creasing rate to produce a rising arterial concentra tion of the tracer approximating a ramp input func tion. This ramp input function serves to delay or to prevent the equilibration of rapidly perfused tissues with the arterial blood during the period of measure ment. Throughout the period of [14C]IAP adminis tration, timed arterial blood samples, freely flowing from the arterial catheter, were collected in glass capillary tubes. The last sample was taken at the time of killing and as long as blood could be with drawn from the arterial catheter. The volume of blood withdrawn was calculated to avoid a nor motensive shock and any change in physiological variables. The rats were killed by decapitation at about 1 min after the beginning of C4C]IAP infusion and brains were removed within I min, frozen in methylbutane chilled to - 40°C, coated with chilled embedding matrix (4% carboxymethylcellulose in water), and stored at - 80°C in plastic bags until sectioned. The content of each capillary tube was trans ferred to a preweighed scintillation vial that was immediately covered and reweighed after blood col lection. The blood samples were then treated with 0.5 ml of tissue solubilizer (Optisolv; FSA Labora tory Supplies) and 0. 5 ml of hydrogen peroxide (30%). The blood concentration of e4C]IAP was then determined by liquid scintillation counting in 10 ml of a scintillation mixture (Optiphase Hisafe; FSA Laboratory Supplies) in a Beckman scintilla tion counter (Model LS 180 1; Beckman Instru ments, Fullerton, CA, U. S. A. ). The concentration of tracer per unit volume of blood in each sample was calculated from the measured amount of 14C,

the weight of the blood sample, and an assumed specific gravity of 1.06 glml for blood. The frozen brains were cut into 20-lJ..m coronal sections at - 22°C in a cryostat. Sections were picked up on glass coverslips and dried on a hot plate (60°C). Sections were autoradiographed on Kodak SB5 film along with calibrated e4C]methyl methacrylate standards (Amersham). All standards were calibrated for their 14C concentration in brain sections, as described previously (Sokoloff et al. , 1977). Adjacent sections were fixed and stained with thionin for histological identification of spe cific nuclei. The autoradiographs were analyzed by quantita tive densitometry with a computerized image processing system (Biocom 200; Les Ulis, France). Optical density measurements for each structure anatomically defined according to the rat's brain atlas of Paxinos and Watson ( 1982) were made bi laterally in a minimum of four brain sections. Tis sue 14C concentrations were determined from the optical densities of the autoradiographic represen tations of the tissues and a calibration curve ob tained from the autoradiographs of the calibrated standards. LCBF values were calculated according to the Fick equation using a brain-blood partition coefficient of 0. 8 (Sakurada et al. , 1978). Physiological variables Just prior to the infusion of [14C]IAP, the mean blood pressure of the animals was measured with an air-damped mercury manometer and the hematocrit value was determined. Arterial pH, Po2, and Pco2 were measured on 40-f.d blood samples by means of a blood gas analyzer (Model 158; Corning, Le Ve sinet, France) just before the onset of the LCBF procedure. Statistical analysis LCBF values were determined in 75 cerebral structures in three groups of rats, one group of con trol and two groups of audiogenic rats, one naive and one kindled. Values of LCBF and of physiolog ical variables in aUdiogenic and kindled rats were compared with those in control animals by means of a Bonferonni's t test for multiple comparisons. In the same test, LCBF values in naive audiogenic an imals were compared with those in kindled audio genic rats. RESULTS Physiological variables As shown in Table 1, physiological variables of audiogenic rats were similar to those of controls, except for Paco2, which was significantly lower by J

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A. NEHLIG ET AL.

262

eas, thalamic anterior and posterior nuclei (Table 5, Fig. 3), forebrain limbic areas such as the basolat eral amygdala, the medial and lateral septum, the dorsal and ventral hippocampus (Table 6, Fig. 3), seven cortical regions, and genu of the corpus cal losum (Table 7, Fig. 3).

TABLE 1. Physiological variables in audiogenic,

kindled and control Wistar AS rats Audiogenic (n = 9)

Control (n = 8)

p.o2 (mm Hg) P.co2 (mm Hg) Arterial pH Arterial blood pressure (mm Hg) Hematocrit (%) Values are means

±

81.5 41.0 7.33

±

128 49

±

± ±

±

7.9 4.7 0.06 6 3

86.8 34.7 7.26

±

126 48

±

± ±

±

7.5 3.3a 0.09 3 3

Kindled

(n

=

7)

80.0 34.5 7.40

±

7.0 2.la 0.05

128 47

±

± ±

±

LCBF rates during kindled audiogenic seizures As shown by the 104% increase in the weighted average value of whole-brain CBF, rates of LCBF were largely increased over control levels in numer ous brain areas of kindled Wistar AS rats. These increases were significant in 68 regions of the 75 studied. LCBF increases involved all structural sys tems studied. There was no change in rates of LCBF compared to control values in the mammil lary body (Table 5), five cortical areas, and genu of the corpus callosum (Table 7). Compared to rats exposed to single audiogenic seizures, rates of LCBF were significantly less in creased in kindled than in naive Wistar AS rats in 33 structures. These were mainly auditory and visual relay nuclei (Table 2, Figs. 2 and 3), the reticular formation and associated areas (Table 3, Fig. 2), six motor regions (Table 4, Fig. 2), and four cortices including the auditory and visual ones (Table 7, Fig. 3). The largest differences between kindled and na ive Wistar AS rats (expressed as percentage of in crease in rates of LCBF over control values) ranged from 167 to 244% and were located in the pontine reticular formation (Table 3, Fig. 2), substantia ni gra, subthalamic nucleus (Table 4, Fig. 2), posterior hypothalamus (Table 5, Fig. 2), and ventral tegmen tal area (Table 6, Fig. 2). The smallest differences (0-50%) were located mostly in hypothalamic and thalamic nuclei (Table 5), limbic and cortical areas, and white matter (Tables 6 and 7, Fig. 3). The ba solateral amygdala was the only brain area in which

5 3

SD of the number of animals in parenthe

ses. ap < 0.01; statistically significant difference from control.

15% in both groups of audiogenic compared to con trol animals. LCBF rates during single audiogenic seizures As reflected by the 156% increase in the weighted average value of whole-brain CBF, rates of LCBF were significantly increased in Wistar AS over con trol levels in all brain areas (Tables 2-7). The high est increases (>250%) were recorded in the vestib ular nucleus (Table 2, Fig. 2), mesencephalic and pontine reticular formation, ventral tegmental area, gigantocellularis nucleus (Table 3, Fig. 2), substan tia nigra pars reticulata and compacta, subthalamic nucleus (Table 4, Fig. 2), dorsomedian, posterior hypothalamus, and parafascicular thalamus (Table 5, Fig. 2). Very marked increases in LCBF (> 180%) were recorded in the inferior and superior colliculi, the lateral geniculate body (Table 2, Fig. 2), the med ullary reticular formation, the locus ceruleus, par vocellular, ambiguus, and spinal trigeminal nuclei (Table 3, Fig. 2), the red nucleus (Table 4), four hypothalamic and two thalamic regions (Table 5, Fig. 2), and the interpeduncular nucleus (Table 6). The structures least affected by audiogenic sei zures, with LCBF rates increased by < 100%, were hypothalamic lateral and magnocellular preoptic ar-

TABLE 2. Effects of audiogenic seizures on LCBF in sensory relay nuclei

Brain structure

Control (n = 8)

Audiogenic (n = 9)

Variation from control

(%)

Kindled

(n =

7)

Variation from control

(%)

Auditory system 32.2 42.2

300.0 426.3

± ±

40.1b 67.9h

38.8 34.5

400.7

±

61.3b

±

±

158.9

±

34.1

413.2 351.0

63.5b 49.4b

83.0 83.1

±

22.6 16.4

246.7 248.6

±

+199

187.8 182.8

±

±

34.4b 36.4b

+197

±

98.1

±

16.1

351.6

±

56.7b

+258

285.0

Medial geniculate body Inferior colliculus

122.1 143.0

±

Lateral lemniscus

148.9 165.8

±

Superior olive Cochlear nucleus Visual system Lateral geniculate body Superior colliculus Vestibular system Vestibular nucleus

±

±

+146 +198 +169 +149 +121

299.7

±

66.2b

239.2 268.4

±

42.0a.d 38.5a.d

281.1 254.3

±

±

+145 +67 +80

32.6a.d 30.3a•c

+60

±

36.0a.c 40.8a.c

+126 +120

±

48.2b

+191

±

+70

Values, expressed as ml 100 g-' min-', are means ± SD of the number of animals in parentheses. ap < 0.05 and bp < 0.01; statistically significant differences from control. cp < 0.05 and dp < 0.01; statistically significant differences from audiogenic rats.

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263

TABLE 3. Effects of audiogenic seizures on LCBF in the reticular formation and associated nuclei

Control (n = 8)

Brain structure Mesencephalic reticular formation Pontine reticular formation Medullary reticular formation Medial raphe Dorsal raphe Locus ceruleus Dorsal tegmentjlm Gigantocellularis nucleus Parvocellularis nucleus Ambiguus nucleus Spinal trigeminal nucleus Nucleus of the solitary tract

Audiogenic (n = 9)

79.0

±

18. 0

±

20.0

289.7 316.7

±

85. 3 69. 1

± ±

92.8

±

16. 8 14. 6 16. 7

198.2 256.7 247.2

±

106.2

±

24.6b 42.7b 33.4b

76.0

±

14. 9

216.1

±

26.8b

110.2

±

21.1

±

58. 2

±

10. 3

274.2 228.2

±

69.0

±

12.0

193.0

±

31.0b 34.0b 21.5b

77. 5 68. 2

±

14. 8

±

30.1b

± ±

12.0 10.4

±

54.9

254.6 203.7 137.0

22.4b 21.7b

± ±

±

34.6b 42.6b

Variation from control (%)

Kindled (n = 7)

+266 +271 +187 +142 +166

173.1 174. 4 148.6 171. 3

±

±

27.2a.d 33.0a.d 26.0b.c 27.3a.c

177.4

±

28.0b.c

+184 +149

160.1

±

23.8a.c

194.8 143. 8

±

30.5a.c 2l.1b.d 11.1a.d

+292

± ±

±

+180 +228 +199

134.0 175.0

±

151. 8

±

+150

103.2

±

±

23.5b.d 29.4b.c 21.8a

Variation from control (%) +119 +104 +115 +61 +91 +111 +77

+147 +94 +126 +123 +88

Values, expressed as ml 100 g-] min -], are means ± SD of the number of animals in parentheses. ap < 0.05 and bp < 0.01; statistically significant differences from control. 'p < 0.05 and dp < 0.01; statistically significant differences from aUdiogenic rats.

LCBF rates were higher during kindled than during single audiogenic seizures (Table 6, Fig. 3). DISCUSSION

changes within seizure networks. All these param eters of cerebral functional activity will be consid ered in the interpretation of our data on LCBF changes induced by single and audiogenic seizures.

The study of cerebral functional activity during seizures can be approached by mUltiple techniques. EEG recordings allow mapping of the cerebral re gions involved in the electrical expression of the seizure and, as is the case in audiogenic seizures, correlate quite well with the behavioral expression of both single and kindled seizures (Marescaux et aI. , 1987; Vergnes et aI. , 1987; Kiessmann et aI. , 1988). The mapping of cerebral metabolism and CBF controls the regional energy cost of the seizure and the eventual mismatch between substrate use and supply. Finally, the expression of early genes such as c-fos allows mapping of the pathways in volved in seizures and may play a critical role in the cascade of molecular events leading to long-lasting

Effects of single audiogenic seizures on rates of LCBF During the occurrence of single audiogenic con vulsions, rates of LCBF were largely increased all over the brain. The most important increases in LCBF, ranging from 180 to 400%, occurred mainly in brain-stem regions as well as in hypothalamic and thalamic areas. The most moderate increases (30140%) occurred in cortical and forebrain regions (Tables 2-7). It has been shown that audiogenic seizures orig inate in brain-stem structures and do not usually involve the forebrain (Browning, 1986; Faingold, 1988; Millan, 1988). Among auditory structures, rates of LCBF are largely increased in the inferior

TABLE 4. Effects of audiogenic seizures on LCBF in motor areas of rats

Brain structure Basal ganglion circuits Dorsomedial caudate nucleus Globus pallidus Substantia nigra pars reticulata Substantia nigra pars compacta Subthalamic nucleus Cerebellar circuits Red nucleus Cerebellar cortex Inferior olive Cerebellar nuclei Dentate nucleus Fastigial nucleus Interpositus nucleus

Control (n = 8) 132.3 53. 1

±

23. 0

±

10.2

57.1

±

8. 6

62. 7 71. 5

±

12.1

±

6.5

86. 2 65. 9

± ±

20. 7 16. 6

98. 2

±

119.2

105.1 108.4

Audiogenic (n = 9) 281.2 144.1 255.3 272.6 349.5

± ± ± ± ±

30.6b 22.2b 47.0b 31. 3b 46.6b

Variation from control (%) +113

232.9 149.3

±

+347 +344

110.9 150.2

±

+388

206.8

±

192.9

±

106.0 188.1

±

27.5a.d 16.5a.c

+124 +61

±

25.8b.c

+92

248.9 241.8 240.4

±

26.2b 35.2b 30.3b

258.4

± ±

28.2b 24.2b

20. 4

±

26.7b

+200 +167 +155

±

19.1

314.2

±

51.4b

+164

±

21. 9 18. 3

283.2 295.3

±

41.1b 38.1b

+169 +172

±

± ±

± ±

34.5b 25.7b 24.2a.d 27.8b.d 27.1b.d


+171

176.0 250.1

±

Kindled (n = 7)

+76

+181 +94

+140 +189

+109 +130 +122

Values, expressed as ml 100 g-] min -], are means ± SD of the number of animals in parentheses. ap < 0.05 and bp < 0.01; statistically significant differences from control. 'p < 0.05 and dp < 0.01; statistically significant differences from audiogenic rats.

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264

TABLE S. Effects of audiogenic seizures on LCBF in the hypothalamus and thalamus

Control (n = 8)

Brain structure Hypothalamus Medial preoptic area Lateral preoptic area Magnocellular preoptic area Anterior Anterolateral Lateral Ventromedian Dorsomedian Posterior Mammillary body Thalamus Anteromedian Anteroventral Ventrolateral Mediodorsal Laterodorsal Ventromedian Reticularis Parafascicular Posterior

60.6 88.7 89.3 64.9 72.2 46.7 43.8 45.7 67.8 126.3

±

110.6 128.8 90.5 97.9 81.7 77.4 69.6 81.2 80.0

±

± ± ± ± ± ± ± ± ±

± ± ± ± ± ± ± ±


Audiogenic (n = 9)

9.6 9.7 15.5 12.2 9.5 6.0 6.8 6.6 11.4 21.0

158.2 174.6 148.5 199.1 219.5 158.9 150.8 166.9 269.2 260.6

±

20.4 23.4 12.3 15.4 21.1 9.8 12.6 18.2 14.5

214.8 287.7 218.5 235.6 218.3 226.1 220.4 311.2 148.5

±

± ± ± ± ± ± ± ± ±

± ± ± ± ± ± ± ±

Kindled (n = 7)

22.7b 23.6a 22.5a 22.3b 24.7b 30.4b 25.6b 26.7b 35.5b 40.6b

+161 +97 +66 +207 +204 +240 +244 +265 +297 +106

134.6 170.5 156.1 176.0 186.3 126.4 129.6 130.5 133.0 160.3

±

18.0b 34.1& 22.3b 26.2b 25.8b 22.9b 26.1b 48.4b 21.5b

+94 +123 +141 +141 +167 +192 +217 +283 +86

232.0 264.5 232.7 274.4 189.9 215.2 182.5 217.0 144.0

±

± ± ± ± ± ± ± ± ±

± ± ± ± ± ± ± ±


23.la 33.8a 37.5a 20.2b 22.4b 30.6b 25.lb 18.9b 26.6b., 23.8'

+122 +92 +75 +171 +158 +171 +196 +186 +96 +27

20.9b 26.8b 38.7b 41.6b 26.2b 30.0b 29.7b 31.2b., 34.7b

+110 +105 +157 +180 +132 +178 +162 +167 +80

Values, expressed as milOO g- \ min -\, are means ± SD of the number of animals in parentheses. ap < 0.05 and bp < 0.01; statistically significant differences from control. 'p < 0.01; statistically significant differences from audiogenic rats.

colliculus. The inferior colliculus is essential for the appearance of audiogenic seizures. Bilateral lesions of the latter structure completely abolish wild run ning and tonic seizures in sound-susceptible rodents (Willot and Lu, 1980; lobe, 198 1; Browning, 1986), whereas electrical stimulation of the inferior collic ulus facilitates sound-induced seizures and may in duce wild running and tonic seizures without expo sure to sound (lobe, 198 1; McCown et aI. , 1984). Electrolytic destruction of the ventral cochlear nu cleus causes only a transient blockade of audiogenic convulsions in rats, and lesions of the medial genic ulate body or the auditory cortex fail to prevent sound-induced seizures. The effects of lesions of

other structures of the auditory pathway such as the superior olive and lateral lemniscus have not been studied (Browning, 1986; Faingold et aI. , 1986). In accordance with these data, increases in LCBF rates in auditory structures were more moderate than in the inferior colliculus during single audio genic seizures in the present study (Table 2). Several brain-stem structures outside of the pri mary auditory pathway have been shown to partic ipate in sound-induced generalized seizures, such as the mesencephalic and pontine reticular forma tion, substantia nigra, and superior colliculus (Browning, 1986; Faingold et aI. , 1986), structures in which rates of LCBF are largely increased in the

TABLE 6. Effects of audiogenic seizures on LCBF in limbic and functionally nonspecific areas

Control (n = 8)

Brain structure

118.4 109.4 92.0 57.9 58.7 71.2 64.5 81.1 62.6 86.7 92.4 120.2 69.4

Accumbens nucleus Medial septum Lateral septum Medial amygdala Central amygdala Basolateral amygdala Dorsal hippocampus Ventral hippocampus Dentate gyrus Interpeduncular nucleus Medial habenula Lateral habenula Ventral tegmental area

± ± ± ± ± ± ± ± ± ± ± ± ±

28.0 22.2 16.1 10.9 10.0 12.8 14.2 18.0 12.3 14.3 28.2 22.3 12.4

-

Audiogenic (n = 9) 309.1 188.7 171.0 143.7 150.3 127.7 125.0 146.9 135.4 255.3 211.8 264.6 258.0

± ± ± ± ± ± ± ± ± ± ± ± ±

32.3b 20.3a 18.8a 28.2b 18.lb 14.8b 14.6b 24.4& 24.4b 28.3b 32.3b 29.5b 37.0b

Variation from control (%) +161 +72 +86 +148 +156 +79 +93 +81 +116 +194 +129 +120 +272

Kindled (n = 7) 261.5 184.5 158.1 148.6 147.2 158.4 113.6 126.3 105.8 196.0 170.9 201.0 132.1

± ± ± ± ± ± ± ± ± ± ± ± ±

20.4b 30.5a 22.4a 42.8b 1O.2b 18.0b.e 16.3' 24.9a 30.4a 27.5b 32.2a 35.8a., 23.0b.d

Variation from control (%) +121 +69 +72 +157 +151 +122 +76 +56 +68 +126 +85 +67 +90

Values, expressed as ml 100 g \ min - \, are means ± SD of the number of animals in parentheses. "p < 0.05 and bp < 0.01; statistically significant differences from control. 'p < 0.05 and dp < 0.01; statistically significant differences from audiogenic rats.

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TABLE 7. Effects of audiogenic seizures on LCBF in the cerebral cortex and white matter areas

Brain structure Cerebral cortex Prefrontal Frontal Anterior cingulate Entorhinal Parietal Motor Olfactory Auditory Visual White matter areas Genu of the corpus callosum Internal capsule Cerebellar white Whole-brain weighted average

Control (n = 8) 195.1 133.0 183.9 53.5 103.0 139.9 135.9 125.7 77.9

±

48.1 44.0 24.6 89.2

±

± ± ± ± ± ± ± ±

± ± ±


Audiogenic (n = 9)

32.3 25.1 28.5 12.8 26.8 16.1 25.1 18.9 20.9

323.5 215.0 347.7 95.9 243.9 228.1 203.7 247.8 186.4

±

6. 1 7.6 6. 1 12.5

6 1.6 97.1 60.0 228.1

±

± ± ± ± ± ± ± ±

± ± ±

Kindled (n = 7)

43.4a 36.3a 45.9b 20.8a 22.1h 28.0b 18.9a 32.2h 28.0b

+66 +62 +89 +79 +137 +63 +50 +97 +139

150.5 182.6 125.8 99.6 199.7 262.5 188.2 144.0 97.1

±

8.8 9.6h 8.2h 29.0b

+28 121 + 143 + 156

50.1 87.6 47.5 181.6

±

+

± ± ± ± ± ± ± ±

± ± ±


32.3c 26.5a 12.5c 16.0a 42.3h 39.3h 48.3 18.4c 24.9c

-23 +37 -32 +86 +94 +88 +38 +15 +25

10.1 9.6h 13.3h 22.6h

+4 +99 +93 +104

Values, expressed as ml lOO g - I min - I , are means ± SO of the number of animals in parentheses. ap < 0.05 and bp < 0.01; statistically significant differences from control. cp < 0.01; statistically significant differences from audiogenic rats.

present study. Large bilateral lesions of the mesen cephalic reticular formation, midbrain, and pontine tegmentum block the tonic and clonic components of audiogenic seizures (Kesner, 1966; Browning et aI. , 1985). The large increase in LCBF recorded in most posterior vegetative nuclei could reflect both the activation of the pontine and medullary reticular formation recorded during aUdiogenic seizures (Browning, 1986; Faingold, 1988; Millan, 1988) and the direct functional activation of these structures occurring during the course of convulsive seizures

for the maintenance of cardiorespiratory and vege tative functions. The substantia nigra is a critical site involved in the control of generalized seizures (McNamara et aI. , 1984; Gale, 1985; Moshe et aI. , 1986; Depaulis et aI. , 1994). In audiogenic rats, lesions of the sub stantia nigra reduce the severity of the seizures (Browning, 1986). The superior colliculus receives afferents from the inferior colliculus and from the substantia nigra and is important in the expression o a

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0 c

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0 fZ W U a:

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SAS 1 KAS

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FIG.2. LCBF increases in structures most sensitive to audio genic seizures, represented as percentage of variation from control values. SAS, single audiogenic seizures; KAS, kin dled audiogenic seizures; IC, inferior colliculus; SC, superior colliculus; MORF, medullary reticular formation; PRF, pon tine reticular formation; MRF, mesencephalic reticular for mation; SNR, substantia nigra pars reticulata; SNC, substan tia nigra pars compacta; VTA, ventral tegmental area; LC, locus ceruleus; AMB, ambiguus nucleus; GIG, gigantocellu laris nucleus; PARV, parvocellularis nucleus; VEST, vestibu lar nucleus; AHY, anterior hypothalamus; VMHY, ventrome dian hypothalamus; PHY, posterior hypothalamus; VMTH, ventromedian thalamus; PTH, parafascicular thalamus. {::rp < 0.05 and {::r{::rp < 0.01, statistically significant differences be tween SAS and KAS.

a: w Q.

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FIG. 3. LCBF increases in structures least sensitive to audio geniC seizures, represented as percentage of variation from control values. SAS, single audiogenic seizures; KAS, kin dled audiogenic seizures; PFT, prefrontal cortex; FT, frontal cortex; OlF, olfactory cortex; CING, cingulate cortex; ENT, entorhinal cortex; MOT, motor cortex; AUO, auditory cortex; lS, lateral septum; MS, medial septum; BlA, basolateral amygdala; OHI, dorsal hippocampus; VHI, ventral hippoca m pus; LP�, lateral preoptic nucleus; POMA, magnocellular preoptic area; AMTH, anteromedian thalamus; GC, genu of the corpus callosum. {::rp < 0.05 and {::r{::rp < 0.01, statistically significant differences between SAS and KAS.

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A. NEHLIG ET AL.

of wild running that initiates aUdiogenic seizures (Cools et aI. , 1984; Faingold et aI. , 1986). Bilateral lesions of the superior colliculus results in the inhi bition of seizures in audiogenic mice (Willot and Lu, 1980). In addition to the structures known to be in volved in the expression of audiogenic seizures, rates of LCBF also increase largely in thalamic and hypothalamic structures (Table 5), motor regions such as both parts of the substantia nigra, and red and subthalamic nuclei (Table 4). The increase in LCBF rates induced by audiogenic seizures in some thalamic and hypothalamic structures and in motor regions in the present study may reflect the involve ment of these structures in various components of seizures elicited by sound stimulation in Wistar AS rats as well as the control of homeostasis and veg etative functions during convulsions. Among forebrain limbic areas, rates of LCBF are not widely increased, except in the accumbens nu cleus and central and medial amygdala (Table 6). The hippocampus and amygdala are particularly seizure-prone areas in the brain and are commonly associated with seizures in a large number of epi lepsy models (Collins et aI. , 1983; Pinard et aI. , 1984; Lothman et aI. , 1985; De Lanerolle et aI. , 1989; Houser et aI. , 1990). However, electrolytic lesions of the amygdala or hippocampus fail to change the intensity of audiogenic seizures accord ing to one study (Kesner, 1966), while other find ings suggest a potential inhibitory role of the hippo campal formation (Kim and Kim, 1962; Reid et aI. , 1983). Sustained e activity on the EEG during a tonic seizure suggests that the hippocampus is in volved in the spread of aUdiogenic seizures during kindling (Hirsch et aI. , 1992). Among the anterior motor regions, LCBF is not widely increased in the caudate nucleus, which, as the hippocampus, plays an inhibitory role in audiogenic seizures (Kesner, 1966). Finally, the neocortex is not involved in au diogenic seizures. Indeed, cortical lesions as well as cortical spreading depression do not affect the ex pression of aUdiogenic seizures (Krushinsky, 1963; Krushinsky et aI. , 1970; lobe, 198 1). Therefore, the rather moderate increase in the rates of LCBF in most forebrain areas is in good accordance with the lack of involvement of these structures in the ex pression of single audiogenic seizures (Browning, 1986; Faingold, 1988; Millan, 1988). In summary, in the present study, there is a good correlation between the nature of the structures in which LCBF rates are mostly increased and the in volvement of those structures in the expression of aUdiogenic seizures. Using qualitative measure ments of 2-e4C]deoxyglucose uptake changes durJ


ing audiogenic seizures in adult GEPR-3, the GEPR with moderate aUdiogenic seizures (lobe et aI. , 1991), Miller et al. ( 1993) found, as in the present study, an activation of brain-stem auditory path ways and the vestibular nucleus, A similar regional mapping of c-fos protein ex pression was obtained during audiogenic seizures. In Wistar AS, a single audiogenic seizure induced the expression of c-fos in the subcortical auditory nuclei and the reticular formation. Few cells were labeled in the amygdala, the thalamic and hypotha lamic nuclei, and the cortex; hippocampal cells were not stained (Simler et aI. , 1994). The same distribution of c-fos labeling was reported in audio genic DBAl2 mice (Le Gal La Salle and Naquet, 1990). Effects of kindled audiogenic seizures on rates of LCBF The most striking difference in the rates of LCBF between kindled and single audiogenic seizures is the general and significant reduction of the increase recorded in the perfusion of most midbrain and hindbrain nuclei (Fig. 2). Conversely, in most fore brain areas, the rate of perfusion of the different structures was moderate and remained similar whether the rats were kindled or not. Only in the basolateral amygdala was LCBF increased in kin dled compared to single audiogenic seizures. It is worth noting that the most marked differ ences in LCBF rates during kindled audiogenic sei zures compared to naive Wistar AS rats are almost specifically located in the regions known to be in volved in the expression of tonic audiogenic sei zures, such as the inferior colliculus, reticular for mation, substantia nigra, and brain-stem vegetative nuclei (Browning, 1986; Faingold, 1988; Millan, 1988). The more moderate increase in LCBF in those brain-stem regions in kindled versus naive Wistar AS is likely to be the reflection of the change in the nature of the seizure. Indeed, kindling of au diogenic seizures reduces the duration or even sup presses the tonic phase of the seizure. The more moderate increases in LCBF recorded in brain-stem regions of kindled than in those of naive Wistar AS rats are also in agreement with the relative decrease in the rates of perfusion after prolonged seizures compared to major early ictal LCBF increases (Meldrum and Nilsson, 1976; Horton et aI. , 1980; Ingvar and Siesj6, 1983; Siesj6 et aI. , 1983; Ingvar et aI. , 1984; Barkai et aI. , 199 1). As a result of kindling, an extension of electrical paroxysmal activity from the brainstem to cortical and forebrain areas has been demonstrated (Mares caux et aI. , 1987; Vergnes et aI., 1987; Kiessman et aI. , 1988). Indeed, in Wistar AS rats, audiogenic


kindling facilitates subsequent hippocampal or amygdala kindling (Hirsch et aI. , 1992). In addition, c-fos expression in the brain stem is not affected by kindling, while it is strongly increased in forebrain areas such as the amygdala, hippocampus, and cor tices (Simler et aI. , 1994). Taken together, all these data demonstrate that audiogenic seizure kindling recruits additional forebrain and, especially, limbic structures into the seizure network. Surprisingly, in the present study, LCBF rates in forebrain struc tures are not widely different in kindled rats com pared to nonkindled animals. The lack of further LCBF stimulation by the repetition of the convul sive event may reflect cerebral adaptative changes. Indeed, electrical kindling increases the number of N-methyl-D-aspartate receptors and changes their pharmacological properties (Nadler et aI. , 1994). Likewise, repeated electroconvulsive seizures have been shown to alter the biochemical parameters of a variety of neurotransmitter systems (Bergstrom and Kellar, 1979; Stockmeier et aI. , 1987). They also induce down-regulation of numerous receptors, such as a- and [3-adrenergic receptors (Bergstrom and Kellar, 1979; Sherwin et aI. , 1990), benzodiaz epine receptors (Bowdler et aI. , 1982), muscarinic receptors (Lerer, 1984), and dopamine autorecep tors (Chiodo and Antelman, 1980). They increase the brain concentration of ),-aminobutyric acid (Bowdler et aI. , 1982) and noradrenaline (Sherwin et aI. , 1990). All these neurotransmitters participate in the regulation of CBF. Therefore, a change in the sensitivity of receptors and/or in the concentra tions, turnover, uptake, and release of neurotrans mitters could attenuate the vasodilatory response of cerebral blood vessels to a repeated stimulation. Nevertheless, the reason the brain appears to react to repeated strong stimulations such as seizures with an attenuation of the LCBF response remains to be elucidated. An additional possibility is that long-term changes induced by seizure repetition could already decrease the basal level of LCBF in kindled compared to naive animals, as shown for glucose utilization in a strain of GEPRs (Saji et aI. , 1993). This aspect will be controlled by further stud ies on naive and kindled Wistar AS rats in the in terictal state. The amygdala is the only forebrain structure in which the LCBF increase during a single seizure was reinforced in kindled rats (Table 6). Likewise, the discrete c-fos labeling in the amygdala following a single audiogenic seizure in naive Wistar AS rats was widely increased after kindling (Simler et aI. , 1994). Therefore, the role of the amygdala in the spread of the sound-induced seizure network from hindbrain to forebrain deserves further attention.

267

The additional question raised by the present study is the use of LCBF to map cerebral functional activity. Indeed, seizure-induced LCBF changes appear to be less sensitive than metabolic changes. In a recent study on pentylenetetrazol-induced sta tus epilepticus in immature rats, we were able to map very specific regional increases, decreases, or lack of change in metabolic activity versus control levels (Pereira de Vasconcelos et aI. , 1992), while regional variations in LCBF were much less spe cific and somewhat different (Pereira de Vasconce los et aI. , 1994). CBF and glucose utilization are not always coupled, especially in pathological situa tions such as seizures, and LCBF is regulated through several factors other than metabolism, as recently shown by the alteration of the relationship between flow and metabolism by a nitric oxide syn thase inhibitor (Macrae et aI. , 1993). However, be cause of the poor temporal resolution of the 2e4C]deoxyglucose technique (about 45 min), the mapping of quantitative metabolic changes during events as short as aUdiogenic seizures, whose du ration does not exceed 60--90 s, is not possible. In conclusion, our data show that there is a good correlation between the brain-stem structures in volved in the expression of single audiogenic sei zures and the highest increases in the rates of LCBF recorded in the present study. After kindling, the more moderate increases in LCBF rates in the brain stem may be related to the evolution of the initially tonic seizure located in the brainstem into tonic clonic seizures also involving the forebrain. How ever, in most forebrain regions, no difference in the rates of LCBF was found between kindled and non kindled animals, despite demonstrated increases in electrical activity and c-fos expression. Whether this apparent mismatch between LCBF and func tional activity reflects adaptative mechanisms due to the repetition of the seizure remains to be eluci dated. REFERENCES Barkai AI, Prohovnik I, Young WL, Durkin M, Nelson HD (1991) Alterations of local cerebral blood flow and glucose uptake by electroconvulsive shock in rats. Bioi Psychiatr 30:269-282 Bergstrom DA, Kellar KJ (1979) Effect of electroconvulsive shock on monoaminergic receptor binding sites in rat brain. Nature 278:464-466 Bowdler JM, Green AR, Minchin MCW, Nutt OJ (1982) Re gional GABA concentration and benzodiazepine receptor number in rat brain following repeated electroconvulsive shock. J Neural Transm 56:3-12 Browning RA, Nelson DK, Mogharreban N, Jobe PC, Laii'd HE II (1985) Effect of midbrain and pontine tegmental lesions on audiogenic seizures in genetically epilepsy-prone rats. Epi lepsia 26:175-183 Browning RA (1986) Neurobiology of seizure disposition-the

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